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Pyrazolines and their pyrazole congeners are important heterocyclic building blocks with numerous applications in the fine chemical industries. However, traditional routes towards these entities are based on multistep syntheses generating substantial amounts of chemical waste. Here we report an alternative approach using UV-light to convert tetrazoles into pyrazolines via a reagent-free photo-click strategy. This route generates nitrile imine dipoles in situ that are trapped with different dipolarophiles rendering a selection of these heterocyclic targets in high chemical yields. A continuous flow method is ultimately realized that generates multigram quantities of product in a safe and readily scalable manner thus demonstrating the value of this photochemical approach for future exploitations in industry.

An efficient continuous flow process is reported for the synthesis of various 1,3,4-oxadiazoles via an iodine-mediated oxidative cyclisation approach. This entails the use of a heated packed-bed reactor filled with solid K2CO3 as a base. Using DMSO as solvent, this flow method generates the target heterocycles within short residence times of 10 minutes and in yields up to 93%. Scale-up of this flow process was achieved (34 mmol/h) and featured an integrated quenching and extraction step. Lastly, the use of an automated in-line chromatography system was exploited to realise a powerful flow platform for the generation of the heterocyclic targets.

Escherichia coli ranks as the number one clinical isolate in the past years in China according to The China Antimicrobial Surveillance Network (CHINET), and its multidrug-resistant (MDR) pathogenic strains account for over 160 million cases of dysentery and one million deaths per year. Here, our work demonstrates that E. coli is highly sensitive to the synergistic combination of SBC3 [1,3-Dibenzyl-4,5-diphenyl-imidazol-2-ylidene silver (I) acetate] and Ebselen, which shows no synergistic toxicity on mammalian cells. The proposed mechanism for the synergistic antibacterial effect of SBC3 in combination with Ebselen is based on directly inhibiting E. coli thioredoxin reductase and rapidly depleting glutathione, resulting in the increase of reactive oxygen species that cause bacterial cell death. Furthermore, the bactericidal efficacy of SBC3 in combination with Ebselen has been confirmed in mild and acute peritonitis mice. In addition, the five most difficult to treat Gram-negative bacteria (including E. coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa) are also highly sensitive to a synergistic combination of SBC3 and Ebselen. Thus, SBC3 in combination with Ebselen has potential as a treatment for clinically important Gram-negative bacterial infections.

Multi-drug resistant bacterial strains (MDR) have become an increasing challenge to our health system, resulting in multiple classical antibiotics being clinically inactive today. As the de-novo development of effective antibiotics is a very costly and time-consuming process, alternative strategies such as the screening of natural and synthetic compound libraries is a simple approach towards finding new lead compounds. We thus report on the antimicrobial evaluation of a small collection of fourteen drug-like compounds featuring indazoles, pyrazoles and pyrazolines as key heterocyclic moieties whose synthesis was achieved in continuous flow mode. It was found that several compounds possessed significant antibacterial potency against clinical and MDR strains of the Staphylococcus and Enterococcus genera, with the lead compound (9) reaching MIC values of 4 µg/mL on those species. In addition, time killing experiments performed on compound 9 on Staphylococcus aureus MDR strains highlight its activity as bacteriostatic. Additional evaluations regarding the physiochemical and pharmacokinetic properties of the most active compounds are reported and showcased, promising drug-likeness, which warrants further explorations of the newly identified antimicrobial lead compound.

A continuous flow process is presented that couples a Curtius rearrangement step with a biocatalytic impurity tagging strategy to produce a series of valuable Cbz-carbamate products. Immobilized CALB was exploited as a robust hydrolase to transform residual benzyl alcohol into easily separable benzyl butyrate. The resulting telescoped flow process was effectively applied across a series of acid substrates rendering the desired carbamate structures in high yield and purity. The derivatization of these products via complementary flow-based Michael addition reactions furthermore demonstrated the creation of β-amino acid species. This strategy thus highlights the applicability of this work towards the creation of important chemical building blocks for the pharmaceutical and speciality chemical industries.

A continuous-flow process is presented that enables the safe generation and derivatization of benzyne under photochemical conditions. This is facilitated by a new high-power LED lamp emitting light at 365 nm. The resulting flow process effectively controls the release of gaseous by-products based on an adjustable backpressure regulator and delivers a series of heterocyclic products in a short residence time of 3 minutes. The robustness of this methodology is demonstrated for the rapid generation of benzotriazoles, 2H-indazoles and various furan-derived adducts, facilitating the preparation of these important heterocyclic scaffolds via a simple and readily scalable flow protocol.

Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

Continuous flow chemistry is by now an established and valued synthesis technology regularly exploited in academic and industrial laboratories to bring about the improved preparation of a variety of molecular structures. Benefits such as better heat and mass transfer, improved process control and safety, a small equipment footprint, as well as the ability to integrate in-line analysis and purification tools into telescoped sequences are often cited when comparing flow to analogous batch processes. In this short review, the latest developments regarding the exploitation of continuous flow protocols towards the synthesis of anticancer drugs are evaluated. Our efforts focus predominately on the period of 2016–2021 and highlight key case studies where either the final active pharmaceutical ingredient (API) or its building blocks were produced continuously. It is hoped that this manuscript will serve as a useful synopsis showcasing the impact of continuous flow chemistry towards the generation of important anticancer drugs.

An assembled suite of flow-based transformations have been used to rapidly scale-up the production of a novel auxin mimic-based herbicide which was required for preliminary field trials. The overall synthetic approach and optimisation studies are described along with a full description of the final reactor configurations employed for the synthesis as well as the downstream processing of the reaction streams.

Background: Association of Ambulatory Blood Pressure Monitoring (ABPM) with mortality depends on cardiac function in hemodialysis patients. Evidence for the predictive power of central Systolic Pressure (cSBP) is inconclusive. Thus, this study aimed to investigate the additional information of ambulatory cSBP in risk prediction in a cohort of hemodialysis patients. Methods: Within the ISAR-study cohort, 344 hemodialysis patients underwent 24 h ABPM on the dialysis day. All-cause and cardiovascular mortality served as endpoints. Risk prediction was performed using Cox regression in patients with or without atrial fibrillation (AF) or heart failure (HF) for peripheral (pSBP) and central systolic pressure calibrated with peripheral systolic and diastolic pressure (cSBP1) or peripheral mean and diastolic pressure (cSBP2). Results: During a mean follow-up of 37.6 (17.5 SD) months, 115 patients died, of whom 47 due to cardiovascular reasons. In patients with AF or HF, a negative association to mortality could be observed, independent of pressure location and calibration (see Table). In patients without AF or HF, these associations were to the opposite directions and cSBP2 was superior to pSBP and cSBP1 for all-cause (pSBP: HR = 1.01, p = 0.30; cSBP1: HR = 1.00, p = 0.77; cSBP2: HR = 1.01, p = 0.06) and cardiovascular (pSBP: HR =1.03, p = 0.02; cSBP1: HR = 1.02, p = 0.06; cSBP2: HR = 1.03, p = 0.003) mortality. This circumstance was confirmed in multivariable analysis combining pSBP and differences between pSBP and cSBP (see Table). Conclusions: This study provides evidence for the additional information of central systolic blood pressure and its dependency on calibration in risk prediction in hemodialysis patients. Further studies are needed to confirm these findings.

The last decade has witnessed a remarkable development towards improved and new photochemical transformations in response to greener and more sustainable chemical synthesis needs. Additionally, the availability of modern continuous flow reactors has enabled widespread applications in view of more streamlined and custom designed flow processes. In this focused review article, we wish to evaluate the standing of the field of continuous flow photochemistry with a specific emphasis on the generation of bioactive entities, including natural products, drugs and their precursors. To this end we highlight key developments in this field that have contributed to the progress achieved to date. Dedicated sections present the variety of suitable reactor designs and set-ups available; a short discussion on the relevance of greener and more sustainable approaches; and selected key applications in the area of bioactive structures. A final section outlines remaining challenges and areas that will benefit from further developments in this fast-moving area. It is hoped that this report provides a valuable update on this important field of synthetic chemistry which may fuel developments in the future.

Objectives: Mortality rate in end-stage renal disease (ESRD) are still at a high level. Sarafidis et al. showed the predictive value of 48h PWV in patients undergoing hemodialysis [1], although recent studies using office measurement showed controversial predictive results. Aim of the present study was to confirm the predictive value of a novel oscillometric measurement of pulse wave velocity on mortality in an elderly cohort of patients with ESRD. Methods: The ISAR study is a prospective and longitudinal study targeting patients with ESRD undergoing hemodialysis. Oscillometric measurement of 24-hour PWV was performed at baseline. Survival analysis included Kaplan-Meier analysis, logrank test and Cox regression. Results: A total of 350 patients had a median age of 69.3 [55.8; 77.3] years. Mean PWV was 9.6 (2.2) m/s and 120 patients died during the mean follow-up of 45 months. PWV was significantly higher in the deceased (10.6 +/− 1.9 m/s) than in surviving patients (9.0 +/− 2.2 m/s). Kaplan-Meier analysis showed differences in dichotomized PWV (cut-off 10 m/s [2]; Logrank test: p = 0.001). For results of univariate Cox regression, see Figure. Adjusted Cox regression analysis showed a significant risk prediction for all-cause mortality (HR 2.322; p = 0.011). Patients older than 50 years showed even higher predictive values (HR 2.442; p = 0.008) as well as patients with PWV values of at least 10 m/s (HR 3.300; p = 0.006). Figure.Univariate hazard-ratios and their 95% confidence intervals for continuous PWV and PWV quartiles (Q1 as reference; ** p = 0.002; *** p < 0.001). Ql: 11.23 m/s. Conclusion: Oscillometric measurement of 24-hour pulse wave velocity is a simple and valid method and has an additional predictive value for all-cause mortality in elderly patients with end-stage renal disease.

The implementation of continuous flow processing as a key enabling technology has transformed the way we conduct chemistry and has expanded our synthetic capabilities. As a result many new preparative routes have been designed towards commercially relevant drug compounds achieving more efficient and reproducible manufacture. This review article aims to illustrate the holistic systems approach and diverse applications of flow chemistry to the preparation of pharmaceutically active molecules, demonstrating the value of this strategy towards every aspect ranging from synthesis, in-line analysis and purification to final formulation and tableting. Although this review will primarily concentrate on large scale continuous processing, additional selected syntheses using micro or meso-scaled flow reactors will be exemplified for key transformations and process control. It is hoped that the reader will gain an appreciation of the innovative technology and transformational nature that flow chemistry can leverage to an overall process.

This note describes a novel reaction cascade in which a tosylhydrazone derivative of eucarvone undergoes a non-classical dimerization process under basic conditions. The key step in this sequence is a dipolar cycloaddition between a diazo species and a transient cyclopropene. A proposed mechanism for this sequence is presented that is supported by single crystal X-ray analysis of the resulting dimer. We believe this unique transformation is of note as it highlights a neat and efficient entry to complex polycyclic architectures containing an embedded pyrazoline moiety.

This note describes a sequence converting an oxime-substituted pyrrolidine into a trisubstituted pyrrole structure. The synthetic route is based on a double chlorination of the pyrrolidine substrate followed by the base induced formation of both an imine and a nitrile oxide functionality. The latter reacts with an immobilized thiourea to yield an isothiocyanate which upon elimination generates the final pyrrole in an unprecedented cascade of events.

Reliable identification of cognitive impairment in hemodialysis patients is of utmost importance, as it is associated with poor outcomes including dialysis withdrawal and death. High prevalence of cognitive impairment has been demonstrated in several studies using brief screening instruments or neuropsychological test batteries. However, the relevance of cognitive impairment as well as the accuracy of screening procedures have never been studied in this patient population.151 chronic hemodialysis patients (mean age 65.78 ± 14.88 years, 73,5% male) underwent cognitive testing under standardized conditions by the Montreal Cognitive Assessment (MoCA) and, in a second step, the Clinical Dementia Rating scale (CDR), an international standard to measure the severity of dementia. For calculating MoCA cut-off values on the basis of the CDR global score, receiver operator characteristics (ROC) analysis and c-statistic were applied.49.0% of patients were categorized as 0.5 in the CDR global with memory being the predominantly affected domain (47.7% of patients scored ≥ 0.5). Youden's Index led to a threshold of 23.5 points for the MoCA test for optimal differentiation between cognitively normal (CDR global < 0.5) and impaired patients (CDR global ≥ 0.5) based on a sensitivity of approximately 99% and a specificity of approximately 74%.Interference of cognitive impairment with patients' independence and daily life was shown using the CDR for the first time in hemodialysis patients. A MoCA score of 23.5 points turned out as optimal threshold to differentiate between patients with and without functional impairment in the CDR, thereby paving the way for implementation of the MoCA test as a quick and thus highly feasible screening instrument for periodic testing in clinical routine.

Robust chemical routes towards valuable bioactive entities such as riboflavines, quinoxalinones and benzodiazepines are described. These make use of modern flow hydrogenation protocols enabling the chemoselective reduction of nitro group containing building blocks in order to rapidly generate the desired amine intermediates in situ. In order to exploit the benefits of continuous processing the individual steps were transformed into a telescoped flow process delivering selected benzodiazepine products on scales of 50 mmol and 120 mmol respectively.

Propargylic amines are important multifunctional building blocks that are frequently exploited in the synthesis of privileged heterocyclic entities. Herein we report on a novel flow process that achieves the safe and effective on-demand synthesis of propargylic amines in a telescoped manner. This process minimizes exposure to hazardous azide intermediates and renders a streamlined route into these building blocks. The value of this approach is demonstrated by the rapid generation of a small selection of drug-like thiazolines that result from a high-yielding reaction cascade between propargylic amines with different aryl isothiocyanates.

Background/Aims: Cigarette smoking is a risk factor for renal damage, but little is known about subclinical effects of smoking on renal hemodynamics and parameters of renal function in humans. We examined the associations of smoking with systemic and renal hemodynamics and renal function parameters in healthy individuals. Methods: Data from 196 potential living kidney donors were analysed retrospectively. Mean arterial blood pressure (MAP), effective renal plasma flow (ERPF) and creatinine clearance had been measured. We additionally calculated parameters of renal hemodynamics. Data were analyzed for the effects of smoking and sex dependent on age and MAP. Results: Systemic and renal hemodynamic parameters did not differ between smokers and non-smokers. In non-smokers of both sexes MAP was negatively correlated with ERPF, and higher MAP was associated with increased renal vascular resistance and with afferent arteriolar resistance, with glomerular pressure (PG) remaining constant. However, in male, but not in female smokers, ERPF and PG increased with MAP. A correlation of age with a steeper decline in ERPF in male smokers was lost in multiple regression analysis. Conclusions: As compared to women, smoking men may exhibit an increased glomerular hydrostatic pressure, which is a known promoter of kidney damage.

This review which is the second in this series summarises the most common synthetic routes as applied to the preparation of many modern pharmaceutical compounds categorised as containing a six-membered heterocyclic ring. The reported examples are based on the top retailing drug molecules combining synthetic information from both scientific journals and the wider patent literature. It is hoped that this compilation, in combination with the previously published review on five-membered rings, will form a comprehensive foundation and reference source for individuals interested in medicinal, synthetic and preparative chemistry.

Isothiocyanates are versatile starting materials for a wide range of chemical reactions. However, their high nucleophilic susceptibility means they are best prepared and used immediately. We report here on a flow platform for the fast and efficient formation of isothiocyanates by the direct conversion of easily prepared chloroximes. To expedite this chemistry a flow insert cartridge containing two immobilised reagents is used to affect the chemical transformation which typically eliminates the requirements for any conventional work-up or purification of the reaction stream.

Introduction: Parameters of arterial stiffness are independent cardiovascular risk factors for end-stage renal disease patients. Significant changes of these parameters between intra- and interdialytic periods have been reported previously [1]. The aim of this cross-sectional study is to describe the influence of the ultrafiltration volume on hemodynamic parameters. Methods: All measurements were obtained with the Mobil-O-Graph 24h PWA (I.E.M. GmbH, Germany) within the ISAR hemodialysis study. Measurement started before the midweek dialysis session and lasted for 24-hours. 348 patients (238 male / 110 female 65 +- 18 years) were included. Intra- and interdialytic parameters were averaged and compared for three subgroups (ultrafiltration volume (UFV) 2000 ml (N = 139)) and all subjects. Results: The results for all patients support the findings of Karpetas et al. [1] (see Table). Beyond [1], the results underpin the differences between subgroups for intra- and interdialytic periods. Furthermore, there are significant differences between intra- and interdialytic periods depending on the ultrafiltration volume (see Table). Exemplarily, there is a significant rise in the augmentation index (26.0 vs. 28.5%, p < 0.05) for UFV > 2000 ml and for central pulse pressure (39.6 vs. 43.4 mmHg and 36.0 vs. 38.3, p < 0.05) for UFV 2000 ml All In Out In Out In Out In Out pSBP (mmHg) 128.9 131.0 123.7 122.3 124.5 123.0 124.8 123.8 pDBP (mmHg) 75.9 74.5 74.9 72.3 75.6 73.1 75.3 * 72.9 pPP (mmHg) 53.0 56.5 48.8 50.0 478.8 49.9 49.4 50.9 HR (bpm) 69.4 70.2 68.1 * 70.8 71.8 73.7 69.8 * 71.9 cSBP (mmHg) 116.9 119.3 112.4 111.9 113.4 112.6 113.4 113.2 cDBP (mmHg) 77.3 75.9 76.3 73.6 77.2 74.6 76.8 * 74.3 cPP (mmHg) 39.6 * 43.4 36.0 * 38.3 36.2 38.0 36.6 ** 38.9 AIx (%) 29.7 31.8 29.5 30.2 26.0 * 28.5 28.1 * 29.8 AIx75 (%) 26.4 28.8 25.5 * 27.7 24.0 ** 27.6 25.1 ** 27.8 PWV (m/s) 10.03 10.14 9.88 9.85 9.08 9.07 9.58 9.58 Abbreviations: peripheral diastolic blood pressure (pDBP), peripheral systolic blood pressure (pSBP), peripheral pulse pressure (pPP), heart rate (HR), central diastolic blood pressure (cDBP), central systolic blood pressure (cSBP), peripheral pulse pressure (pPP), augmentation index (AIx, AIx75) and pulse wave velocity (PWV) */**marks a significant difference between intra- and interdialytic periods (p < 0.05 and p < 0.01, respectively). Table:Averaged hemodynamic parameters for intra- and interdialytic periods (In vs. Out) for different subgroups based on ultrafiltration volume (UF) and all subjects.

Introduction: Heart rate variability (HRV) analysis is a non-invasive tool for assessing the cardiac health. There is evidence that the cardiac autonomic system and central hemodynamics respond to hemodialysis. The aim of this work is to compare HRV parameters and augmentation index (AIx) between intra- and interdialytic periods in patients with end-stage renal disease. Methods: All 24-h electrocardiogram data were obtained using the Lifecard CF digital Holter recorder (Delmar Reynolds/Spacelabs, Germany) and 24-h pulse wave analysis (PWA) measurements were taken with the Mobil-O-Graph 24h PWA (I.E.M. GmbH, Germany) within the ISAR hemodialysis study. Two-hundred patients (132 men / 68 women: 61+−16 years) were included. HRV was analyzed in the time- and frequency-domain in 5-min segments. AIx values and HRV parameters were averaged for intra- and interdialytic periods. Results: The low to high frequency ratio (LF/HF) as a representative of sympathovagal balance (2.85 vs 3.36, p

This review presents a comprehensive overview on selected synthetic routes towards commercial drug compounds as published in both journal and patent literature. Owing to the vast number of potential structures, we have concentrated only on those drugs containing five-membered heterocycles and focused principally on the assembly of the heterocyclic core. In order to target the most representative chemical entities the examples discussed have been selected from the top 200 best selling drugs of recent years.

Introduction: There are known issues and ongoing discussions regarding calibration methods using peripheral mean and diastolic or peripheral systolic and diastolic pressure for the determination of central systolic blood pressure. The aim of this cross-sectional study is an analysis of determinants for the differences in central systolic blood pressure between the two calibration methods. Methods: All measurements were obtained with the Mobil-O-Graph 24h PWA monitor calibrated with both calibration methods within the ISAR hemodialysis study. Measurement started after a short dialysis interval prior to dialysis and lasted for 24-hours. Peripheral and central systolic blood pressures were averaged. 345 patients (231 male / 114 female; 65 +− 15 years) were included and grouped according to peripheral systolic blood pressure and one of the following factors: age, sex, body-mass-index, diabetes mellitus, length of dialysis and coronary artery disease. Results: There is a significant influence of age, diabetes mellitus, and coronary artery disease, but not of sex, body-mass-index and length of dialysis. Exemplarily, significant differences in central systolic blood pressure were 12.5 +− 7.3 (pSBP 130 mmHg, no diabetes mellitus) and 26.1 +− 16.2 (pSBP > 130 mmHg, diabetes mellitus). Conclusions: These findings enhance our understanding of the differences in the two calibration methods. These results are generally assumed to play a role in future risk classification. It is suggested that the association of these factors is investigated in future studies.

This short note describes a one-step synthesis of the title compound from commercially available starting materials and reports its full spectroscopic characterization data.

Background: Although low magnesium levels have been associated with an increased mortality in dialysis patients, they are kept low by routinely-used dialysates containing 0.50 mmol/L magnesium. Thus, we investigated the impact of a higher dialysate magnesium concentration on mortality. Methods: 25 patients on high dialysate magnesium (HDM) of 0.75 mmol/L were 1:2 matched to 50 patients on low dialysate magnesium (LDM) of 0.50 mmol/L and followed up for 3 years with regards to all-cause and cardiovascular mortality. Patients were matched according to age, gender, a modified version of the Charlson Comorbidity Index (CCI), and smoking status. Results: During the follow-up period, five patients died in the HDM and 18 patients in the LDM group. Patients in the HDM group had significantly higher ionized serum magnesium levels than matched controls (0.64 ± 0.12 mmol/L vs. 0.57 ± 0.10 mmol/L, p = 0.034). Log rank test showed no difference between treatment groups for all-cause mortality. After adjustment for age and CCI, Cox proportional hazards regression showed that HDM independently predicted a 65% risk reduction for all-cause mortality (hazard ratio 0.35, 95% confidence interval [CI]: 0.13, 0.97). Estimated 3-year probability of death from a cardiovascular event was 14.5% (95% CI: 7.9, 25.8) in the LDM group vs. 0% in the HDM group. Log rank test found a significant group difference for cardiovascular mortality (χ2 = 4.15, p = 0.042). Conclusions: Our data suggests that there might be a beneficial effect of an increased dialysate magnesium on cardiovascular mortality in chronic dialysis patients.

Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.

The synthesis of diazonium salts is historically an important transformation extensively utilized in dye manufacture. However the highly reactive nature of the diazonium functionality has additionally led to the development of many new reactions including several carbon-carbon bond forming processes. It is therefore highly desirable to determine optimum conditions for the formation of diazonium compounds utilizing the latest processing tools such as flow chemistry to take advantage of the increased safety and continuous manufacturing capabilities. Herein we report a series of flow-based procedures to prepare diazonium salts for subsequent in-situ consumption.